The grant application proposes continuation of research toward two long term objectives: understanding 1) the translocation mechanism of the conserved viral DNA packaging motor and its regulation; and 2) the evolution of the internal proteins injected to counter novel DNA translocating restriction enzymes. The packaging motor consists of the T4 large terminase subunit working at the portal dodecamer of the prohead to translocate dye labeled short DNAs of any sequence with near 100% efficiency. Following single complexes we will analyze portal-GFP prohead packaging. By FCS and nuclease assay short nicked DNAs are not packaged, unlike bubble mismatch DNAs, suggesting that the motor introduces torsion into the DNA to translocate it. FRET will be used to establish with double dye DNAs that the dimensions of the DNA are changed by the stalled motor. Additionally FRET between and within tagged terminase and portal will establish distance and conformational changes during packaging. FRET will also be used to establish the proximity of the two ends in packaged heads. We will determine the headful cutting specificity of the terminase. We will complete our work showing the participation of the late sigma factor (gp55)-sliding clamp (gp45) complex in packaging initiation in vivo on nicked circular DNAs by large terminase subunit gp17 interaction with a number of regulatory proteins. In the second objective, a diverse family of CTS-injected proteins has evolved by gene expansion at the IPI locus to challenge a novel family of NTP driven DNA translocating restriction enzymes. Our ongoing analysis of two of the gmrS/gmrD restriction endonucleases (CT and UT), two polymorphic phage gene antagonists (IPI* and IP5* whose structures we have determined), and diverse target sugar HMC adducts of these enzymes reveals an evolutionary pathway generating complexity on a nanoscale. Overall, effort devoted to the first DNA packaging objectives will be more heavily weighted than to the second restriction endonuclease-injected inhibitors portion, the two research objectives of our previous grant. However, approaches to studying DNA translocation in the two research areas will be complementary.